The present invention relates to an arrangement for detecting changes of a magnetic response with at least one magnetic particle provided with an external layer in a carrier fluid, thus the method comprises using a measuring method comprising measuring the characteristic rotation time of said magnetic particle with respect to said external layer, which measuring method involves measuring Brownian relaxation in said carrier fluid under the influence of an external alternating magnetic field.
Magnetic spherical particles with a diameter of less than about 20 nm are magnetic mono domains both in a magnetic field and in the zero field. A particle being a magnetic mono domain means that the particle only contains one magnetization direction.
Depending on the size, geometry, temperature, measurement time, magnetic field and material of the particles, they can either be thermally blocked or super paramagnetic. The direction of the magnetization for thermally blocked particles are oriented in a specific direction in the magnetic particle in proportion to the crystallographic orientation of the particle, and xe2x80x9clockedxe2x80x9d to this direction, meanwhile studying the particle system. Under influence of an external magnetic field, the entire particle physical rotates so that their magnetization directions gradually partly coincide with the direction of the external added field.
Small magnetic particles can be manufactured in a number of materials, for example magnetite (Fe3O4), maghemite (xcex3-Fe2O4), cobalt doped iron oxide or cobalt iron oxide (CoFe2O4). Other magnetic materials, especially (but not exclusively) rare earth metals (for example ytterbium or neodymium), their alloys or compounds containing rare earth metals, or doped magnetic (element) substances can also be possible. The sizes of the particles that can be produced from about 3 nm to about 30 nm. The final size in this process depends on a number of different parameters during manufacturing.
Magnetization in small particles can relax in two different ways, via Nxc3xa9elian relaxation or on the other hand via Brownian relaxation. These relaxation phenomena are related to particles with a magnetic arranged structure. They should not be mistaken for nuclear magnetic (NMR) resonance phenomenon""s, the latter describes resonances within the atomic nucleus. The latter resonance phenomena have resonance frequencies typically within the GHz-range unlike resonance frequencies for the phenomenon considered in this patent, which are in the range of a few Hz to several MHz.
Nxc3xa9elian Relaxation
In Nxc3xa9elian relaxation the magnetization in the particle relax without the particle physical rotating (no thermal blocking). The relaxation period for this kind of relaxation strongly depends on size, temperature, material and (at high particle concentrations) on the magnetic interaction between the particles. For this relaxation being available the magnetization direction in the particle has to change direction fast in time, the particles have to be super-paramagnetic. Nxc3xa9elian relaxation period in the zero field can be described according the equation below:       τ    N    =            τ      0        ⁢          ⅇ              KV        kT            
wherein xcfx840 is a characteristic relaxation period, K is the magnetic anisotropic constant, V magnetic particle volume, k is Boltzman""s constant and T temperature.
Brownian Relaxation
In the Brownian relaxation, the magnetization-direction rotates when the particle physically rotates. For this relaxation being available the magnetization has to be locked in a specific direction in the particle, and the particle has to be thermal blocked. The relaxation period for Brownian relaxation depends on hydrodynamic particle volume, viscosity of the carrier fluid in which the particles are dispersed in, contact between the surface of the particle and the fluid layer nearest it""s surface (Hydrophobic and hydrophilic respectively). The Brownian relaxation can approximately be described according to the equation below:       τ    B    =            3      ⁢              V        H            ⁢      η        kT  
wherein VH is the hydrodynamic volume for the total particle (inclusive of the polymer layer), xcex7 viscosity for the surrounding carrier fluid, k is Boltzmann""s constant and T is the temperature. The above derivation assumes a perfect wetting (hydrophilic) has been assumed and a constant rotation speed (the initial approximation has been neglected).
The Brownian relaxation period accordingly depends on the (effective) size of the particle and the environmental effect on the particle. To discern if a particle shows Brownian relaxation or Nxc3xa9elian relaxation you can among other things study whether external influences (for example a different fluid viscosity, temperature changes, applied static magnetic field) changes the relaxation period.
You can also study the phenomenon in the frequency domain, which involves determining the resonance frequencies regarding the particle system. These can be obtained for example by means of AC-susceptometry (for Brownian relaxation some Hz till kHz region and for Nxc3xa9elian relaxation typically in the MHz region).
As is apparent from the above, a Brownian movement (Brownian relaxation) depends, among other things on the volume of the particle: the lager particle the longer relaxation period that is and the smaller the movement of the particle gets. Relaxations periods for particles lager than about 1 xcexcm are much longer than 1 second, which in practice means a negligible movement. Even these particles, though can be used at detection. Larger particles can, however, show other types of relaxations wherein the inertia of the particles and visco-elastic characteristics of the carrier fluid must be included for a sufficient data interpretation.
Frequency Susceptibility
The magnetization for a particle system in an alternating magnetic field can be described according to:
M="khgr"H=("khgr"xe2x80x2xe2x88x92j"khgr"xe2x80x3)H
wherein M is the magnetization, H the alternating external magnetic field, "khgr" is the frequency dependent complex susceptibility consisting of an in phase component (real part), "khgr"xe2x80x2, and one out of phase component (imaginary part), "khgr"xe2x80x3. The in phase and the out of phase components for a magnetic particle system can approximately be described as:                     χ        xe2x80x2            =                        χ          0                                      1            +                                          (                                  2                  ⁢                  π                  ⁢                                      xe2x80x83                                    ⁢                  f                  ⁢                                      xe2x80x83                                    ⁢                  τ                                )                            2                                ⁢                      xe2x80x83                                ⁢          
                  χ      xe2x80x2xe2x80x2        =                            χ          0                ⁡                  (                      2            ⁢            π            ⁢                          xe2x80x83                        ⁢            f            ⁢                          xe2x80x83                        ⁢            τ                    )                                      1          +                                    (                              2                ⁢                π                ⁢                                  xe2x80x83                                ⁢                f                ⁢                                  xe2x80x83                                ⁢                τ                            )                        2                          ⁢                  xe2x80x83                    
wherein "khgr"0 is the DC value of the susceptibility and xcfx84 is the relaxation period for magnetic relaxation.
Assuming a particle system with varying particle sizes wherein some of the particles go through Brownian relaxation (the larger particles) and some Nxc3xa9elian relaxation (the smaller particles) you obtain a magnetic response contribution from both the relaxation processes depending on the frequency range AC field. FIG. 1 shows schematically the total magnetic response as a function of the frequency for the particle system that shows both Brownian and Nxc3xa9elian relaxation. The upper curve (dashed line) in the figure is the real part of the susceptibility and the lower curve (continuous line) is the imaginary part of the susceptibility. The maximum for the imaginary part at lower frequencies is from the Brownian relaxation and the maximum at high frequencies is from the Nxc3xa9elian relaxation. The total magnetic response is the sum of the contributions from both the processes for both real and imaginary part of the susceptibility.
For this application only the Brownian relation is interesting, therefore the discussion is concentrated at these lower frequencies.
For a particle system with particles showing Brownian relaxation with only one hydrodynamic volume you obtain a maximum in the out of phase component ("khgr"xe2x80x3, the imaginary part of the complex susceptibility) at a frequency according to:       f    max    =            1              2        ⁢                  πτ          B                      =          kT              6        ⁢        π        ⁢                  xe2x80x83                ⁢                  V          H                ⁢        η            
Around this frequency, ƒmax, the real part of the susceptibility, "khgr"xe2x80x2, will decline very much while the imaginary part of the susceptibility, "khgr"xe2x80x3, will exhibit a maximum. The value of "khgr"xe2x80x3 at the maximum (B in the FIG. 1) is among other things a measure of the number of particles that goes through Brownian relaxation while the level of the magnetic response for "khgr"xe2x80x2 (C in FIG. 1) after the maximum in "khgr"xe2x80x3 is a measure of the total number of particles that still magnetically can follow the applied AC field (in this case particles that goes through Nxc3xa9elian relaxation). At sufficient low frequencies all particles can magnetically follow the AC field, that is, the real part of the susceptibility at these low frequencies (A in FIG. 1) is a measure of the total number of particles. The contribution from the Brownian particles can then be quantified as the difference between the total contribute, A and the Nxc3xa9elian contribution, C (D in FIG. 1). At higher frequencies, a new maximum is obtained in "khgr"xe2x80x3 as a result of the Nxc3xa9elian relaxation (E in FIG. 1). The comparison between these two values is therefore a measure of the concentration of particles in a sample that goes through the Brownian relaxation, which is of interest for this application. The width of the maximum of "khgr"xe2x80x3, xcex4 fmax (and the speed of the subside of "khgr"xe2x80x2) is a measure of energy dissipation due to the fluids repercussion on the particles (the friction). The friction vary with (above all) the spreading in the hydrodynamic volume between the particles as a particle population in a sample can show., but also depends partly also on statistical (temperature dependent) fluctuations.
By measuring susceptibility, the Brownian relaxation and the energy dissipation, one can determine the total concentration of particles, the degree of particles that goes through Brownian relaxation in this particle population, the mean size of a particle in a carrier fluid and the spreading in particle volumes.
Magnetic particles have earlier been used as carrier of bio-molecules or antibodies for measuring changes in their magnetic response. In these methods, the particles are either bound to a fixed surface or the particles are aggregated. One measures how the magnetic resonance decrease with time after the particle system is magnetized, see R. Kxc3x6titz, T. Bunte, W. Weitschies, L. Trahms, Superconducting quantum interference device-based magnetic nanoparticle relaxation measurement as a novel tool for the binding specific detection of biological binding reactions, J. Appl. Phys., 81, 8, 4317, 1997, or one measures the magnetic response when an external magnetic field is applied over the magnetic particles, see K. Enpuku, T. Minotani, M. Hotta, A. Nakohado, Application of High Tc SQUID Magnetometer to Biological Immunoassays, IEEE Transactions on Applied Superconductivity, Vol. 11, No. 1, 661-664, 2001. In performing these measurements, one distinguishes between the Nxc3xa9elian relaxation and the Brownian relaxation. The measurements are performed with a totally different technique then that used for the present invention, so called SQUID-technique that requires the use of cryofluids and advanced electronics. H. L. Grossman, Y. R. Chemla, Y. Poon, R. Stevens, J. Clarke, and M. D. Alper, Rapid, Sensitive, Selective Detection of Pathogenic Agents using a SQUID Microscope, Eurosensors XIV, 27-30, 2000, also uses antibody cased magnetic nanoparticles for determining specific target molecules, but combines this with the SQUID technologyxe2x80x94that is, with a superconducting detector.
There are three substantially differences between the procedure according to present invention and the above mentioned methods:
(i) the physical principles behind the measurements according to the invention are different from earlier performances when others have chosen to measure in time/period domains instead of in frequency domains as shown in this case, and also that the it is necessary to xe2x80x9cpre-magnetizesxe2x80x9d the particle system.
(ii) The method of measurement that many uses for measuring is constructed from a very sensitive, but expensive and complicated technology,xe2x80x94namely the SQUID technology.
(iii) The invention is based on that the agglomeration of the particles. This is accomplished through providing the particles with a surface having characteristics that prevent agglomerations from being formed. For example, monoclonal antibodies reacting specifically with the substance to be analyzed can cover the surface of the particles. According to known techniques, bio molecules with multiple ways of bonding have been analyzed.
In Kxc3x6titz, H. Matz, L. Trahms, H. Koch, W. Weitschies, T. Rheinlander, W. Semmler, T. Bunte, SQUID based remanence measurements for immunoassays, IEEE Transactions on Applied Superconductivity, vol. 7, No. 2,3678-81, 1997, the Brownian relaxation in a system of magnetic nanoparticles has also been studied. They have been using magnetic balls covered with biotin. To this system they have added different amounts of avidin. When avidin has 4 bonding places to biotin, avidin-including agglomerate is created. In the present method, the molecule 1 and molecule 2 are chosen in such a way that no agglomerate is created. For example, monoclonal antibodies (molecule 1) can be bonded to the magnetic ball. This monoclonal antibody bonds to a specific etipop on the target molecule, which leads to prevention of agglomerate (FIG. 9).
Yet, another thing that distinguish the method according to the invention and similar methods is that in this case how the frequency dependent of the magnetic response is changed at different measurement frequencies using a relatively simple measuring arrangement. What further distinguishes the present method is that, according to the invention different bio-molecules or antibodies are bonded to the particle surface that changes the hydrodynamic volume. According to earlier methods particles are bond to a fixed surface or the particles are aggregated.
U.S. Pat. No. 6,027,946 describes a process for magneto-relaxometric quantitative detection of analyte in the fluid and solid phase, substances for magnetorelaxometric detection of analyses and immuno-magnetography. The process uses SQUID technique and both the device and the method differ from the present invention.
The invention relates to detecting changes in the magnetic response of the magnetic particles that shows the Brownian relaxation in a carrier fluid (for example water or a suitable buffer fluid, or another fluid suitable for the bio-molecules that are the final target for the detection) under influence of an external AC-magnetic field. Upon modification of the efficient volume of the particles or their interaction with the surrounding fluid, for example when bio-molecules or antibodies are bond on their surfaces, the hydrodynamic volume of respective particles will be changes (increase) which involves a change (reduction) of the frequency, ƒmax, wherein the out of phase component of the magnetic susceptibility is at a maximum.
Hence, the initially mentioned method comprises use of a method further involving, upon modification of the effective volume of the particle or its interaction with the carrier fluid, a change in the hydrodynamic volume of the particle, which implies a change of the frequency where an out of phase component of the magnetic susceptibility is at a maximum. The measurement is actually a relative measurement in which changes in a modified particle system are compared with an original system. At least two sample containers and two detector coils are used for the measurement. Preferably, an oscillator circuit at a frequency is used, that frequency being the resonance frequency, wherein the detector coils are placed as a frequency-determining element in the oscillator circuit so that they are out of phase with each other. The frequency change and/or effect or the amplitude of the oscillations from the oscillation circuit over the coils is therefore measured.
An external oscillator/frequency generator can be arranged, in which the coils are in an alternating bridge so that the difference between both detector coils are measured, and so that the phase difference between the output current and/or voltage of the frequency generator and a current/voltage over the bridge is measured. In this case an amplitude difference between the oscillator output current/voltage can be measured and compared with an amplitude of the current/voltage in the bridge. The measurement is accomplished at one or several different frequencies.
A noise source can be used as well and that the response of the system can be analyzed by means of a FFT (Fast Fourier Transform) analysis of an output signal.
According to one embodiment, the signal difference is set to zero between the coils, which is done through mechanically adjusting position of the respective sample containers, and alternatively changing the position of the respective detection coils so that the difference signal is minimized. The zero setting can be achieved by minimizing the signal through adding a determined amount of a magnetic substance in one of the spaces wherein the sample containers are placed, so that the substance creates an extra contribution to the original signal that therefore can be set to zero. The magnetic substance shows substantially zero magnetic loss (imaginary part=0) and that the real part of the susceptibility is constant in the examined frequency range.
The method is preferably but not exclusively used in analysis instruments for analyzing different bio-molecules or other molecules in fluid. The molecules, comprises one or several proteins in a fluid solution, such as blood, blood plasma, serum or urine. The analysis (molecule 2) can be connected to the particle through interaction with a second molecule (molecule 1), which is connected to the particle before the analysis starts. Molecules that can be integrated specifically which each other can comprise one or more of antibodyxe2x80x94antigen pairs, receptorxe2x80x94hormone pairs, two complementary single strings of DNA and enzymexe2x80x94substrate/enzymexe2x80x94inhibitor pairs.
According to a preferred embodiment, the surface of the magnetic particle is modified through covering the surface with one or more of dextrane, with alkanethiols with suitable end groups or with some peptides. The dextrane surface (or another suitable intermediate layer) can then be bonded to a first molecule, for example an antibody, be bond by means of, for example cyanobromide activation or carboxyl acid activation.
The invention also relates to an arrangement for performance of a method for detection of changes in the magnetic response of at least one magnetic particle provided with an external layer in a carrier fluid, the method comprising measuring the characteristic rotation period of the magnetic particles with respect to the agitation of the external layer. The arrangement comprises at least two substantially identically detection coils connected to detection electronics and sample containers for absorbing carrier fluid. An excitation coil can surround the detection coils and sample containers for generation of a homogeneous magnetic field at the sample container. According to one embodiment, the excitation coil, measurement coils and sample container are placed concentrically and adjusted around its vertical center axis. The arrangement can furthermore comprise an oscillator system wherein the detection coils constitutes the frequency-determining element in an oscillator circuit. The coils are arranged in the oscillator return coil. The respective coils that surround the samples are electrically phase shifted versus each other so that the resonance frequency is determined from the difference between the inductance and the resistance of the respective coil. The coils are placed in an AC-bridge. Additionally, an operation amplifier can be arranged to subtract two voltages from each other.
The arrangement comprises a phase locking circuit in one embodiment. In another embodiment the arrangement comprises oscillator/frequency generator signal to generate a time variable current to excite the coils by means of white noise. Frequency-depending information is received through an FFT-filtering of the response.
The inventions also relates to a method of determining an amount of molecules in a carrier fluid containing magnetic particles, where the determination may comprise the steps of:
A. providing the magnetic particles with a layer, which interacts/reacts with the substance to be analyzed;
B. compounding the magnetic particles with a sample to be analyzed with respect to molecules,
C. filling a sample container with the fluid being prepared according to step B,
D. placing sample container in the detection system,
E. applying an external measure field over the sample with a certain amplitude and frequency,
F. measuring up the magnetic response (both in phase and out of phase components) at this frequency,
G. changing frequency and executing the measurement according to steps D and E,
H. analyzing the result through determining a Brownian relaxation period from in phase and out of phase components through using data in the examined frequency interval.
The method further involves determining the frequency shift (for same value of in phase and out of phase components) at different frequencies. The molecule consists of a bio molecule.